A Guide to Bacteria Preservation:
Refrigeration, Freezing and Freeze Drying

Between stock cultures, mutant strains, and genetically engineered
variants, the number of individual bacterial cultures which any one lab
can accumulate can be numerous. Indeed, the number of variations
created in the process of engineering one plasmid can be astounding.
And most labs will hold on to all those and other variations as you'll
never know what you might need tomorrow. Consequently, preserving
all those bacterial cultures and genetic variants is something to be
approached with thought.

A bacterial culture in a capped tube is in a closed environment.
Though the culture may start healthy, given time the number of viable
cells will decrease to zero. The goal of preserving the cultures is
to slow that death rate so that when the culture is revisited, some of
the cells are still viable and available for culturing. The reasons
the cells die can be numerous, but in every instance are based on the
inherent chemistry of the cells and their environment. If the
deleterious chemical reactions can be slowed or halted, then the overall
culture will remain viable for a longer period of time.

There are two basic approaches to slowing the rate of deleterious
reactions in a culture of bacteria. The first is to lower the
temperature which decreases the rate of all chemical reactions.
This can be done using refrigerators, mechanical freezers, and liquid
nitrogen freezers. The second option is to remove water from the
culture, a process which can be tricky and involves sublimation of water
using a lyophilizer.

Following is a brief discussion of the major options for preserving
bacteria. The strengths and weakness of each option is reported.

Refrigeration

Bacteria can survive for a short period of time at 4°C.
For strains that are used daily or weekly, cultures grown on agar slants
or plates can be stored in a refrigerator assuming that precaution has
been taken to avoid contamination. Cultures should be prepared
using standard techniques and then sealed before storing. For
slants, we recommend using screw capped tubes. For cultures on
Petri dishes, the plates need to be sealed with Parafilm. Sealing
the plates not only helps to prevent molds from sneaking into the plates,
but it slows the agar from drying. For anything over a week or two,
cultures can be stored as stabs in small, flat-bottomed screw capped
vials. In this technique, vials are filled with a small amount of
agar medium (e.g., 1 ml) and sterilized. Bacteria are then
introduced into the solidified agar with a sterile needle. The
culture is incubated overnight with loose caps and then stored at 4°C
with tight caps. Cultures stored in stabs are more resistant to
drying and contamination, but they will lose viability more quickly than
frozen stocks. The length of time a stab can remain viable is
dependent upon the strain. Some manuals claim that stabs are good
for a year however it is unwise to make that assumption unless it is
tested.

Freezing

Freezing is a good way to store bacteria. Generally, the colder
the storage temperature, the longer the culture will retain viable cells.
Freezers can be split into three categories: laboratory, ultralow,
and cryogenic. The problem faced by bacteria (and other cells)
stored in freezers is ice crystals. Ice can damage cells by
dehydration caused by localized increases in salt concentration. As
water is converted to ice, solutes accumulate in the residual free water
and this high concentration of solutes can denature biomolecules.
Ice can also rupture membranes, though this problem is more often
associated with cells lacking walls, such as cultured animal cells.
To lessen the negative effects of freezing, glycerol is often used as a
cryoprotectant. Glycerol is produced by many fish and insects to
defend against cold temperatures by depressing the freezing point of the
cells, enhancing supercooling, and by protection from ice. With
bacteria, adding glycerol to final concentration of 15% will help to keep
cells viable under all freezing conditions (seethis link for a protocol
or this link for aready to use freezing tube). The following are some
specifics for each freezer category.

Laboratory freezers
are those that can pull temperatures down to -20 to -40°C.
These are single stage systems (one compressor) and often called general
purpose freezers. Bacteria can be stored for moderate periods of
time, e.g., 1 year, in general purpose freezers. It is best to use
freezers without frost-free temperature cycling as this can wreak havoc
on cells and other temperature sensitive biomolecules. General
purpose freezers are inexpensive and found in most labs, thus they are
readily available for storing cultures. The downside is that they
are not sufficiently cold for long-term storage.

Ultralow freezers
are two stage systems (two compressors each having a different
refrigerant) which pull down to around -86°C.
Ultralow freezers are very prevalent, but space in them can sometimes be
limited and competitive. Ultralow freezers also are much more
expensive to purchase, run and maintain. The upside is that cells
stored at -80°C tend to remain
viable for several years. The lower temperature generated by
ultralow freezers substantially reduces chemical reactions within the
culture. However, molecular motion still occurs in frozen cells and
thus the viability of the culture will decline. It is important to
regularly monitor cultures to assess their level of viability.

Cryogenic freezers
are very cold and rely on liquid nitrogen or specialized mechanical
systems to operate. For biological samples, cryogenic storage
should be below -130°C. At
this temperature, the molecular motion of water is halted and cells are
trapped in a glass-like matrix. Bacteria stored in cryogenic
freezers retain their viability for many years. In our laboratory
bacterial and yeast cultures have been maintained at -140°C
for 15 years without significant loss of viability. Storing cells
in cryogenic freezers is the most effective and, as compared to freeze
drying, the easiest method for long-term storage. The downside is
cost and potential vulnerability of stocks to power outages, mechanical
failures, and failed deliveries of liquid nitrogen. Additionally,
tubes should never be stored in tanks submersed in liquid nitrogen.
Screw cap tubes leak and will pull the nitrogen into the tube along with
contaminants (
see link for more information). Liquid nitrogen vapor phase
freezers will effectively avoid this problem, but these freezers are very
expensive (upwards of $10K) and require large volumes of liquid nitrogen.
An alternative is mechanical cryogenic freezers that can go as low as
-150°C, but these are also very
expensive to purchase (about $20K). Both cryogenic freezers will
cost several hundred dollars a month to operate.

Freeze Drying

In an aqueous system, such as a living cell, water not only serves as
the medium for enzymatic reactions, but also spontaneous negative
reactions such as free radical formation. Removing water halts both
enzymatic and non-enzymatic reactions. Freeze drying is one method
of removing this water. Many bacteria can be preserved very
effectively by freeze drying. By freezing the cells in a medium
that contains a lyoprotectant (usually sucrose) and then pulling the
water out using a vacuum (sublimation), cells can be effectively
preserved. This method is laborious and requires specialized
equipment, but it has the advantage of generating stock cultures that are
unaffected by power outages and empty liquid nitrogen tanks.
Furthermore, if cultures are routinely shipped to other labs, freeze
dried cultures do not require special handling. The downside on
freeze drying is that not all cultures react the same way thus some
experimentation is required to optimize the process for each strain.
For any lab which is serious about producing and maintaining a culture
collection, then freeze drying should be included as a major method for
preservation.